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Journal of Enzyme Inhibition and Medicinal Chemistry
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Aryl derivatives of 3H-1,2-benzoxathiepine
2,2-dioxide as carbonic anhydrase inhibitors
Aleksandrs Pustenko , Alessio Nocentini , Anastasija Balašova , Ahmed
Alafeefy , Mikhail Krasavin , Raivis Žalubovskis & Claudiu T. Supuran
To cite this article:
Aleksandrs Pustenko , Alessio Nocentini , Anastasija Balašova , Ahmed
Alafeefy , Mikhail Krasavin , Raivis Žalubovskis & Claudiu T. Supuran (2020) Aryl derivatives of
3H-1,2-benzoxathiepine 2,2-dioxide as carbonic anhydrase inhibitors, Journal of Enzyme Inhibition
and Medicinal Chemistry, 35:1, 245-254, DOI: 10.1080/14756366.2019.1695795
To link to this article: https://doi.org/10.1080/14756366.2019.1695795
© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Published online: 02 Dec 2019.
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RESEARCH PAPER
Aryl derivatives of 3H-1,2-benzoxathiepine 2,2-dioxide as carbonic
anhydrase inhibitors
Aleksandrs Pustenko
a,b, Alessio Nocentini
c, Anastasija Bala
sova
a, Ahmed Alafeefy
d, Mikhail Krasavin
e,
Raivis
Zalubovskis
a,band Claudiu T. Supuran
ca
Latvian Institute of Organic Synthesis, Riga, Latvia;bInstitute of Technology of Organic Chemistry, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Riga, Latvia;cDipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Florence, Italy;dFaculty of Pharmacy, University Technology MARA, UiTM, Bandar, Malaysia;eChemistry Department, Saint Petersburg State University, Saint Petersburg, Russian Federation
ABSTRACT
A new series of homosulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) possessing various substitution patterns and moieties in the 7, 8 or 9 position of the heterocylic ring were prepared by original proce-dures and investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the human (h) hCA I, II, IX and XII. The 8-substituted homosulfocoumarins were the most effective hCA IX/XII inhibitors followed by the 7-substituted derivatives, whereas the substitution pattern in position 9 led to less effective binders for the transmembrane, tumour-associated isoforms IX/XII. The cytosolic isoforms hCA I and II were not inhibited by these compounds, similar to the sulfocoumarins/cou-marins investigated earlier. As hCA IX and XII are validated anti-tumour targets, with one sulphonamide (SLC-0111) in Phase Ib/II clinical trials, finding derivatives with better selectivity for inhibiting the tumour-associated isoforms over the cytosolic ones, as the homosulfocoumarins reported here, is of cru-cial importance. ARTICLE HISTORY Received 7 October 2019 Revised 13 November 2019 Accepted 14 November 2019 KEYWORDS Carbonic anhydrase; transmembrane isoforms; sulfocoumarin; homosulfo-coumarin; isoform-selective inhibitor
1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes wide-spread in nature, being encoded by at least eight different genetic families, which have been identified in organisms all over the phylogenetic tree1–3. By catalysing a crucial physiologic reaction, by which CO2 is hydrated with the formation of a weak base
(bicarbonate) and a strong acid (hydronium ions), these enzymes are involved in a multitude of physiologic processes, starting with pH regulation and ending with metabolism1,3–6. As thus, CAs are drug targets for decades, with their inhibitors having pharmaco-logical applications in a multitude of fields1,3–5. The primary sul-phonamides were discovered as CA inhibitors (CAIs) in the 40 s, and most of the drugs that were launched in the next decades as diuretics, antiepileptics, or antiglaucoma agents targeting CAs belonged to this class of compounds1,3–5. Although highly effect-ive as CAIs1, the sulphonamides generally indiscriminately inhibit most a-CA isoforms present in mammals (at least 15 in humans, and 16 in other vertebrates1) as well as CAs belonging to the other genetic families (b-, c-, d-, f-, g-, h- and i-CAs)2–5and for this reason alternative CAI classes were searched for. In fact, in the last 10 years, a multitude of new chemotypes as well as novel CA inhibition mechanisms were reported1,4,7–9, which highly enriched our understanding of these enzymes and also allowed for obtain-ing isoform-selective CAIs targetobtain-ing all the mammalian iso-forms4,7–9. Among the new such chemotypes, which also showed the highest levels of isoform selectivity, were the coumarins9, the sulfocoumarins7,8 and their congeners, homosulfocoumarins
(3H-1,2-benzoxathiepine 2,2-dioxides)10. Considering the fact that this last chemotype was only recently reported and rather poorly investigated10, we report here a series of new aryl-3H-1,2-benzoxa-thiepine 2,2-dioxides substituted in various positions of the het-erocyclic ring, which have been designed in order to explore the chemical space around this new CA inhibitory chemotype and to see whether the presence of various moieties in position 7, 8 or 9 of the heterocyclic system maintains the desired enzyme inhibi-tory activity and selectivity for the target isoforms.
2. Materials and methods
2.1. ChemistryReagents, starting materials and solvents were obtained from commercial sources and used as received. Thin-layer chromatog-raphy was performed on silica gel, spots were visualised with UV light (254 and 365 nm). Melting points were determined on an OptiMelt automated melting point system. IR spectra were recorded on Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Bruker Advance Neo (400 MHz) spec-trometer with chemical shifts values (d) in ppm relative to TMS using the residual DMSO-d6 signal (
1
H 2.50; 13C 39.52) or CDCl3
signal (1H 7.26;13C 77.16) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a mass spectrometer with a Q-TOF micro mass analyser using the ESI technique. Elemental analyses were measured using Carlo Erba (EA1108) apparatus (Milan, Italy).
CONTACTClaudiu T. Supuran claudiu.supuran@unifi.it Neurofarba Department, University of Florence, Firenze 50121, Italy
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 2020, VOL. 35, NO. 1, 245–254
2-Hydroxy-5-iodobenzaldehyde (2)
To a solution of salicylaldehyde (1) (8.73 mL, 81.9 mmol) in AcOH (40 mL) iodine monochloride (4.92 mL, 98.3 mmol) was added11. Reaction mixture was stirred 24 h at 40C, then cooled to r.t. EtOH (60 mL) was added and all volatiles were removed in vac-uum. CH2Cl2 (60 mL) and water (100 mL) were added, the phases
were separated and the aqueous phase was extracted with CH2Cl2
(3 50 mL). The combined organic phases were washed with 10% Na2S2O3(1 60 mL), brine (1 60 mL), dried over Na2SO4, filtered
and concentrated. The residue was purified by column chromatog-raphy on silica gel (PE/EtOAc 3:1), the crude product was re-crys-tallised from EtOH to afford product 2 (17.1 g, 84%) as yellowish solid.1H NMR (400 MHz, DMSO-d6) d¼ 6.85 (d, 1H, J ¼ 8.6 Hz), 7.77
(dd, 1H, J¼ 8.6, 2.4 Hz), 7.87 (d, 1H, J ¼ 2.4 Hz), 10.16 (s, 1H), 10.92 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6) d¼ 81.4, 120.1, 124.6,
136.7, 144.1, 160.3, 189.8 ppm.
Prop-2-ene-1-sulphonyl chloride (4)
Compound was synthesised using previously described procedure by our group10. To a solution of Na2SO3(30.2 g; 0.24 mol) in water
(140 mL) ally bromide (17.4 mL; 0.20 mol) was added and the reac-tion mixture was refluxed overnight. After cooling to room tem-perature, reaction mixture was washed with Et2O (3 50 mL).
Aqueous phase was concentrated. Crude white solid was dried under high vacuum at 100C for 6 h. To the white solid at 0C POCl3 (120 mL) was added, and mixture was refluxed for 4 h. After
cooling to room temperature dry THF (60 mL) was added and reaction mixture was vigorously stirred for 10 min and filtered. Filter cake was suspended in dry THF (60 mL), suspension was vig-orously stirred for 10 min and filtered. Filtrates were combined and solvent was carefully driven off on rotary evaporator. Residue was distilled in vacuum (10 mbar) and fraction with boiling point 38–42C was collected, to give prop-2-ene-1-sulfonil chloride (4) as colourless oil (18.6 g, 66%), which was used in further reactions without additional purification.
General procedure for the synthesis of ethenylphenoles (3, 14, 18, 27)
To a stirred solution of methyltriphenylphosphonium bromide (2.60 eq) in dry THF (5 mL/1 mmol of methyltriphenylphosphonium bromide), was added tBuOK (3.2 eq) in several portions over 20 min. Reaction mixture was stirred for 1 h at r.t. Corresponding benzaldehyde (1 eq) was added and stirring continued at room temperature for 24 h. Reaction mixture was diluted with CH2Cl2
(4 mL/1 mmol of methyltriphenylphosphonium bromide). Organic layer was washed with water (2 20 mL) and brine (2 20 mL), and dried over Na2SO4, filtered and concentrated. The crude
prod-uct was purified by column chromatography on silica gel (PE/EtOAc 4:1).
4-Iodo-2-ethenylphenol (3)
Compound 3 was prepared according to the general procedure from methyltriphenylphosphonium bromide (14.98 g, 37.0 mmol), t-BuOK (5.79 g, 51.6 mmol) and 2-hydroxy-5-iodobenzaldehyde (2) (4.00 g, 16.1 mmol) as yellowish solid (3.29 g, 83%)12. 1H NMR
(400 MHz, DMSO-d6) d¼ 5.23 (dd, 1H, J ¼ 11.3, 1.4 Hz), 5.80 (dd, 1H, J¼ 17.8, 1.4 Hz), 6.67 (d, 1H, J ¼ 8.6 Hz), 6.77–6.87 (m, 1H), 7.38 (dd, 1H, J¼ 8.5, 2.3 Hz), 7.70 (d, 1H, J ¼ 2.3 Hz), 9.94 (s, 1H) ppm 13 C NMR (100 MHz, DMSO-d6) d¼ 81.4, 115.1, 118.4, 126.9, 130.4, 134.4, 137.0, 154.6 ppm. 3-Bromo-2-ethenylphenol (14)
Compound 14 was prepared according to the general procedure from methyltriphenylphosphonium bromide (18.48 g; 51.7 mmol), t-BuOK (7.15 g; 63.7 mmol) and 2-bromo-5-hydroxybenzaldehyde (13) (4.00 g, 19.9 mmol) as yellowish solid (3.25 g; 82%). 1H NMR (400 MHz, DMSO-d6) d¼ 5.51 (dd, 1H, J ¼ 12.0, 2.4 Hz), 6.06 (dd, 1H, J¼ 17.7, 2.4 Hz), 6.76 (dd, 1H, J ¼ 17.7, 11.9 Hz), 6.86–6.91 (m, 1H), 6.98 (t, 1H, J¼ 8.0 Hz), 7.07 (dd, 1H, J ¼ 8.0, 1.2 Hz), 10.18 (s, 1H) ppm13C NMR (100 MHz, DMSO-d6) d¼ 115.4, 120.9, 123.2, 123.4, 124.2, 129.1, 132.2, 157.1 ppm. 5-Bromo-2-ethenylphenol (18)
Compound 18 was prepared according to the general procedure from methyltriphenylphosphonium bromide (18.48 g; 51.7 mmol), t-BuOK (7.15 g; 63.7 mmol) and 4-bromo-2-hydroxybenzaldehyde (17) (4.00 g, 19.9 mmol) as yellowish solid (3.01 g; 76%)13.1H NMR (400 MHz, DMSO-d6) d¼ 5.24 (dd, 1H, J ¼ 11.3, 1.6 Hz), 5.79 (dd, 1H, J¼ 17.8, 1.6 Hz), 6.86 (dd, 1H, J ¼ 17.8, 11.3 Hz), 6.93–6.97 (m, 1H), 7.00 (d, 1H, J¼ 2.0 Hz), 7.37 (d, 1H, J ¼ 8.3 Hz), 10.13 (s, 1H) ppm 13C NMR (100 MHz, DMSO-d6) d¼ 114.6, 118.3, 120.8, 122.0, 123.5, 128.0, 130.8, 155.7 ppm. 2-Bromo-6-ethenylphenol (27)
Compound 27 was prepared according to the general procedure from methyltriphenylphosphonium bromide (18.48 g; 51.7 mmol), t-BuOK (7.15 g; 63.7 mmol) and 3-bromo-2-hydroxybenzaldehyde (26) (4.00 g, 19.9 mmol) as yellowish solid (3.17 g; 80%)14.
1 H NMR (400 MHz, DMSO-d6) d¼ 5.29 (dd, 1H, J ¼ 11.2, 1.3 Hz), 5.78 (dd, 1H, J¼ 17.6, 1.4 Hz), 6.80 (t, 1H, J ¼ 7.8 Hz), 7.02 (dd, 1H, J¼ 17.6, 11.2 Hz), 7.41–7.49 (m, 2H), 9.32 (s, 1H) ppm 13C NMR (100 MHz, DMSO-d6) d¼ 112.2, 115.5, 121.3, 125.4, 127.5, 131.4, 132.1, 150.7 ppm.
General procedure for diolefine (5, 15, 19, 28) synthesis
To a stirred solution of corresponding ethenylphenol (3, 14, 18, 27) (1 eq) in CH2Cl2 (10 mL/1 mmol corresponding ethenylphenol)
at 0C was added prop-2-ene-1-sulphonyl chloride (4) (1.39 eq) and Et3N (1.4 eq). Reaction mixture was stirred overnight (20 h) at
room temperature. Water (30 mL) was added, reaction mixture was extracted with EtOAc (3 40 mL), combined organic extracts were washed with brine (2 40 mL), and dried over dried over Na2SO4, filtered and concentrated. The crude product was purified
4-Iodo-2-ethenylphenyl prop-2-ene-1-sulfonate (5)
Compound 5 was prepared according to the general procedure from 4-iodo-2-ethenylphenol (3) (2.00 g; 8.13 mmol), prop-2-ene-1-sulphonyl chloride (4) (1.11 mL; 10.57 mmol) and NEt3 (1.58 mL;
11.38 mmol) as yellowish oil (2.36 g; 83%). IR (film, cm1) max¼
1373 (S¼ O), 1160 (S ¼ O). 1H NMR (400 MHz, DMSO-d6)
d¼ 4.46–4.50 (m, 2H), 5.44–5.55 (m, 2H), 5.56–5.63 (m, 1H), 5.85–5.97 (m, 1H), 6.02 (d, 1H, J ¼ 17.6 Hz), 6.84 (dd, 1H, J ¼ 17.8, 11.2 Hz), 7.15 (d, 1H, J¼ 8.6 Hz), 7.72 (dd, 1H, J ¼ 8.6, 2.2 Hz), 8.10 (d, 1H, J¼ 2.2 Hz) ppm. 13C NMR (100 MHz, DMSO-d6) d¼ 54.9,
93.0, 119.0, 124.6, 125.0, 125.3, 128.4, 133.1, 134.9, 137.9, 145.7 ppm. HRMS (ESI) [Mþ H]þ: m/z calcd for C11H12O3SI:
350.9552. Found 350.9542.
3-Bromo-2-vinylphenyl prop-2-ene-1-sulfonate (15)
Compound 15 was prepared according to the general procedure from 3-bromo-2- ethenylphenol (14) (2.00 g; 10.05 mmol), prop-2-ene-1-sulphonyl chloride (4) (1.37 mL; 13.06 mmol) and NEt3
(1.96 mL; 14.07 mmol) as yellowish oil (2.01 g; 66%). IR (film, cm1) max¼ 1368 (S ¼ O), 1174 (S ¼ O), 1160 (S ¼ O).
1H NMR (400 MHz, DMSO-d 6) d¼ 4.41 (dt, 2H, J ¼ 7.2, 1.0 Hz), 5.49–5.53 (m, 1H), 5.55–5.61 (m, 1H), 5.69–5.76 (m, 2H), 5.83–5.94 (m, 1H), 6.63 (dd, 1H, J¼ 17.9, 11.7 Hz), 7.30–7.35 (m, 1H), 7.43–7.46 (m, 1H), 7.67 (dd, 1H, J ¼ 8.0, 1.1 Hz) ppm. 13C NMR (100 MHz, DMSO-d6) d¼ 55.4, 122.4, 123.5, 123.7, 124.5, 125.2, 129.8, 130.6, 131.6, 131.9, 146.9 ppm. HRMS (ESI) [Mþ H]þ: m/z calcd for C11H12O3SBr: 302.9691. Found 302.9681.
5-Bromo-2-vinylphenyl prop-2-ene-1-sulfonate (19)
Compound 19 was prepared according to the general procedure from 5-bromo-2- ethenylphenol (18) (2.00 g; 10.05 mmol), prop-2-ene-1-sulphonyl chloride (4) (1.37 mL; 13.06 mmol) and NEt3
(1.96 mL; 14.07 mmol) as yellowish oil (1.65 g; 54%). IR (film, cm1) max¼ 1377 (S ¼ O), 1161 (S ¼ O). 1H NMR (400 MHz, DMSO-d6)
d¼ 4.54 (dt, 2H, J ¼ 7.2, 1.0 Hz), 5.48 (dd, 1H, J ¼ 11.2, 0.8 Hz), 5.52–5.56 (m, 1H), 5.58–5.64 (m, 1H), 5.86–5.98 (m, 1H), 5.99 (dd, 1H, J¼ 17.6, 0.9 Hz), 6.89 (dd, 1H, J ¼ 17.8, 11.2 Hz), 7.55–7.59 (m, 2H), 7.73–7.77 (m, 1H) ppm. 13C NMR (100 MHz, DMSO-d 6) d¼ 55.1, 118.4, 120.8, 124.5, 125.4, 125.6, 128.1, 128.7, 130.3, 130.5, 146.1 ppm. HRMS (ESI) [Mþ H]þ: m/z calcd for C11H12O3SBr:
302.9691. Found 302.9684.
2-Bromo-6-vinylphenyl prop-2-ene-1-sulfonate (28)
Compound 28 was prepared according to the general procedure from 2-bromo-6-ethenylphenol (27) (2.00 g; 10.05 mmol), prop-2-ene-1-sulphonyl chloride (4) (1.37 mL; 13.06 mmol) and NEt3
(1.96 mL; 14.07 mmol) as yellowish oil (2.62 g; 86%). IR (film, cm1) max¼ 1367 (S ¼ O), 1179 (S ¼ O), 1165 (S ¼ O).1H NMR (400 MHz,
CDCl3) d¼ 4.31 (dt, 2H, J ¼ 7.2, 1.0 Hz), 5.45 (dd, 1H, J ¼ 11.0,
0.80 Hz), 5.57–5.65 (m, 2H), 5.81 (dd, 1H, J ¼ 17.5, 0.8 Hz), 6.03–6.15 (m, 1H), 7.07–7.17 (m, 2H), 7.52–7.59 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3) d¼ 57.9, 117.7, 118.4, 123.9, 125.7, 126.0, 128.3,
130.9, 133.1, 135.1, 144.4 ppm. HRMS (ESI) [Mþ H]þ: m/z calcd for C11H12O3SBr: 302.9691. Found 302.9681.
General method for 3H-1,2-benzoxathiepine 2,2-dioxide halogen derivative (7, 20, 29) synthesis
To a solution of corresponding diolefine (5, 15, 19, 28) (1.0 eq) in dry, degassed toluene (15 mL/1 mmol corresponding diolefine) ruthenium catalyst 6 (5 mol %) was added. Reaction mixture was bubbled with argon for 5 min and sealed, stirred at 70C for 4 h. After cooling to r.t. it was concentrated, and the crude product was purified by column chromatography on silica gel (EtOAc/PE 1:4). Products were re-crystallised from EtOH.
7-Iodo-3H-1,2-benzoxathiepine 2,2-dioxide (7)
Compound 7 was prepared according to the general procedure from diolefine (5) (1.00 g; 2.86 mmol) and ruthenium catalyst 6 (0.14 g; 0.14 mmol) as yellowish solid (0.82 g; 89%). Mp 127–128C. IR (film, cm1)max¼ 1370 (S ¼ O), 1164 (S ¼ O), 1155 (S ¼ O). 1H
NMR (400 MHz, DMSO-d6) d¼ 4.52 (dd, 2H, J ¼ 5.8, 1.3 Hz),
5.97–6.04 (m, 1H), 6.82–6.87 (m, 1H), 7.14 (d, 1H, J ¼ 8.5 Hz), 7.79 (dd, 1H, J¼ 8.5, 2.2 Hz), 7.88 (d, 1H, J ¼ 2.2 Hz) ppm. 13C NMR (100 MHz, DMSO-d6) d¼ 51.6, 92.3, 121.5, 124.5, 129.8, 130.4,
138.7, 139.6, 146.7 ppm. Anal. Calcd for C9H7IO3S: C, 33.56; H, 2.19.
Found: C, 33.55; H, 2.21.
8-Bromo-3H-1,2-benzoxathiepine 2,2-dioxide (20)
Compound 20 was prepared according to the general procedure from diolefine (19) (1.23 g; 4.06 mmol) and ruthenium catalyst 6 (0.19 g; 0.20 mmol) as white solid (1.0 g; 90%). Mp 144–145C. IR (film, cm1) max¼ 1359 (S ¼ O), 1182 (S ¼ O), 1165 (S ¼ O). 1H
NMR (400 MHz, DMSO-d6) d¼ 4.54 (dd, 2H, J ¼ 5.8, 1.0 Hz),
5.95–6.05 (m, 1H), 6.87 (d, 1H, J ¼ 11.4 Hz), 7.42–7.47 (m, 1H), 7.58–7.66 (m, 2H) ppm. 13C NMR (100 MHz, DMSO-d6) d¼ 51.9,
120.9, 122.0, 125.2, 127.5, 130.1, 130.3, 133.0, 147.1 ppm. Anal. Calcd for C9H7BrO3S: C, 39.29; H, 2.56. Found: C, 39.28; H, 2.59.
9-Bromo-3H-1,2-benzoxathiepine 2,2-dioxide (29)
Compound 29 was prepared according to the general procedure from diolefine (28) (2.20 g; 7.26 mmol) and ruthenium catalyst 6 (0.34 g; 0.36 mmol) as yellowish solid (1.55 g; 78%). Mp 113–114C. IR (film, cm1) max¼ 1364 (S ¼ O), 1177 (S ¼ O).
1 H NMR (400 MHz, CDCl3) d¼ 4.10 (dd, 2H, J ¼ 6.0, 1.2 Hz), 5.95–6.03 (m, 1H), 6.82–6.87 (m, 1H), 7.18 (t, 1H, J ¼ 7.8 Hz), 7.24–7.28 (m, 1H), 7.66 (dd, 1H, J¼ 7.9, 1.6 Hz) ppm. 13C NMR (100 MHz, CDCl3) d¼ 51.8, 117.7, 120.1, 128.0, 130.0, 130.1, 132.2, 134.2, 144.9 ppm. Anal. Calcd for C9H7BrO3S: C, 39.29; H, 2.56. Found: C, 39.28;
H, 2.58.
General method for 3H-1,2-benzoxathiepine 2,2-dioxide aril derivative (8–12, 21–25 and 30–34) synthesis
In a pressure tube corresponding 3H-1,2-benzoxathiepine 2,2-diox-ide halogen derivative (7, 20, 29) (1.0 eq) was dissolved in dry toluene (6 mL/1 mmol corresponding 3H-1,2-benzoxathiepine 2,2-dioxide halogen derivative), degassed water was added (5% from toluene volume), corresponding boronic acid (1.5 eq), K3PO4 (2.0
eq) and Pd(PPh3)4 (0.1 eq). Reaction mixture was bubbled with
argon 5 min, tube was sealed and heated for 16 h at 100C tem-perature. Reaction mixture was cooled to r.t., filtered through cel-lite was washed with EtOAc (40 mL). Mixture was evaporated and crude product was purified by column chromatography on silica gel (EtOAc/PE 1:3). Products were re-crystallised from EtOH.
7- Phenyl-3H-1,2-benzoxathiepine 2,2-dioxide (8)
Compound 8 was prepared according to the general procedure from 7-iodo-3H-1,2-benzoxathiepine 2,2-dioxide (7) (0.20 g; 0.62 mmol) phenylboronic acid (0.11 g; 0.93 mmol), K3PO4 (0.26 g;
1.24 mmol) and Pd(PPh3)4 (72 mg; 0.062 mmol) as white solid
(95 mg; 56%). Mp 144–145 C. IR (film, cm1) max¼1366 (S ¼ O),
1363 (S¼ O), 1172 (S ¼ O), 1164 (S ¼ O).1H NMR (400 MHz, CDCl3)
d¼ 4.06 (dd, 2H, J ¼ 6.2, 0.8 Hz), 5.99–6.07 (m, 1H), 6.95 (d, 1H, J¼ 11.0 Hz), 7.38–7.43 (m, 2H), 7.44–7.52 (m, 3H), 7.54–7.58 (m, 2H), 7.62 (dd, 1H, J¼ 8.4, 2.2 Hz) ppm.13C NMR (100 MHz, CDCl3)
d¼ 51.4, 119.8, 123.3, 127.3, 128.1, 128.5, 129.1, 129.3, 129.4, 132.9, 139.4, 140.6, 147.1 ppm. Anal. Calcd for C15H12O3S: C, 66.16;
H, 4.44.Found: C, 66.06; H, 4.45.
7–(4-Methoxyphenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (9)
Compound 9 was prepared according to the general procedure from 7-iodo-3H-1,2-benzoxathiepine 2,2-dioxide (7) (0.20 g; 0.62 mmol) 4-methoxyphenylboronic acid (0.14 g; 0.93 mmol), K3PO4 (0.26 g; 1.24 mmol) and Pd(PPh3)4 (72 mg; 0.062 mmol) as
yellowish solid (115 mg; 61%). Mp 162–163 C. IR (film, cm1)max¼
1395 (S¼ O), 1375 (S ¼ O), 1179 (S ¼ O), 1156 (S ¼ O). 1H NMR
(400 MHz, DMSO-d6) d¼ 3.80 (s, 3H), 4.50 (dd, 2H, J ¼ 5.8, 1.0 Hz),
5.98–6.06 (m, 1H), 6.97 (d, 1H, J ¼ 11.2 Hz), 7.02–7.07 (m, 2H), 7.38 (d, 1H, J¼ 8.4 Hz), 7.62–7.67 (m, 2H), 7.69 (dd, 1H, J ¼ 8.4, 2.4 Hz), 7.73 (d, 1H, J¼ 2.4 Hz) ppm. 13C NMR (100 MHz, DMSO-d6)
d¼ 51.6, 55.2, 114.5, 120.5, 122.7, 127.9, 128.0, 128.4, 129.0, 130.8, 131.2, 138.7, 145.8, 159.3 ppm. Anal. Calcd for C16H14O4S: C, 63.56;
H, 4.67. Found: C, 63.38; H, 4.68.
7–(4-Fluorophenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (10)
Compound 10 was prepared according to the general procedure from 7-iodo-3H-1,2-benzoxathiepine 2,2-dioxide (7) (0.20 g; 0.62 mmol) (4-fluorophenyl)boronic acid (0.13 g; 0.93 mmol), K3PO4
(0.26 g; 1.24 mmol) and Pd(PPh3)4 (72 mg; 0.062 mmol) as white
solid (79 mg; 44%). Mp 117–118C. IR (film, cm1) max¼1373
(S¼ O), 1181 (S ¼ O), 1168 (S ¼ O). 1H NMR (400 MHz, CDCl3)
d¼ 4.06 (dd, 2H, J ¼ 6.2, 1.2 Hz), 5.99–6.06 (m, 1H), 6.93 (d, 1H, J¼ 11.0 Hz), 7.12–7.18 (m, 2H), 7.39 (d, 1H, J ¼ 8.4 Hz), 7.45 (d, 1H, J¼ 2.3 Hz), 7.49–7.55 (m, 2H), 7.57 (dd, 1H, J ¼ 8.4, 2.3 Hz) ppm. 13 C NMR (100 MHz, CDCl3) d¼ 51.4, 116.1 (d, J ¼ 21.6 Hz), 119.9, 123.4, 128.6, 128.9, 129.0, 129.2, 129.3, 132.8, 135.6 (d, J¼ 3.4 Hz), 139.6, 147.1, 163.0 (d, J¼ 247.0 Hz) ppm. Anal. Calcd for C15H11FO3S: C, 62.06; H, 3.82. Found: C, 62.34; H, 3.83.
7–(4-(Trifluoromethyl)phenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (11)
Compound 11 was prepared according to the general procedure from 7-iodo-3H-1,2-benzoxathiepine 2,2-dioxide (7) (0.20 g; 0.62 mmol) (4-(trifluoromethyl)phenyl)boronic acid (0.18 g; 0.93 mmol), K3PO4 (0.26 g; 1.24 mmol) and Pd(PPh3)4 (72 mg;
0.062 mmol) as white solid (140 mg; 66%). Mp 166–168C. IR (film, cm1) max¼1357 (S ¼ O), 1332 (S ¼ O), 1166 (S ¼ O). 1 H NMR (400 MHz, DMSO-d6) d¼ 4.56 (dd, 2H, J ¼ 5.8, 1.0 Hz), 6.00–6.08 (m, 1H), 6.99 (d, 1H, J ¼ 11.4 Hz), 7.47 (d, 1H, J ¼ 8.4 Hz), 7.81–7.86 (m, 3H), 7.88 (d, 1H, J ¼ 2.2 Hz), 7.94 (d, 2H, J ¼ 8.2 Hz) ppm. 13C NMR (100 MHz, DMSO-d 6) d¼ 51.8, 120.8, 123.0, 124.3 (q, J¼ 273.0 Hz), 125.9 (q, J ¼ 3.7 Hz), 127.7, 128.3 (q, J ¼ 32.0 Hz), 128.6, 128.9, 130.3, 130.8, 137.4, 142.5, 146.9 ppm. Anal. Calcd for C16H11F3O3S: C, 56.47; H, 3.26. Found: C, 56.46; H, 3.28.
7–(4-(Ethoxycarbonyl)phenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (12)
Compound 12 was prepared according to the general procedure from 7-iodo-3H-1,2-benzoxathiepine 2,2-dioxide (7) (0.20 g; 0.62 mmol) (4-(ethoxycarbonyl)phenyl)boronic acid (0.18 g; 0.93 mmol), K3PO4 (0.26 g; 1.24 mmol) and Pd(PPh3)4 (72 mg;
0.062 mmol) as yellowish solid (96 mg; 44%). Mp 141–142C. IR (film, cm1)max¼ 1701 (C ¼ O), 1380 (S ¼ O), 1184 (S ¼ O), 1170
(S¼ O). 1 H NMR (400 MHz, DMSO-d6) d¼ 1.34 (t, 3H, J ¼ 7.1 Hz), 4.34 (q, 2H, J¼ 7.1 Hz), 4.55 (dd, 2H, J ¼ 5.8, 1.2 Hz), 6.00–6.08 (m, 1H), 6.99 (d, 1H, J¼ 11.5 Hz), 7.46 (d, 1H, J ¼ 8.5 Hz), 7.83 (dd, 1H, J¼ 8.5, 2.3 Hz), 7.85–7.90 (m, 3H), 8.03–8.08 (m, 2H) ppm.13C NMR (100 MHz, DMSO-d6) d¼ 14.2, 51.7, 60.8, 120.8, 123.0, 127.1, 128.6, 128.8, 129.2, 129.8, 130.1, 130.8, 137.7, 142.9, 146.9, 165.4 ppm. Anal. Calcd for C18H16O5S: C, 62.78; H, 4.68. Found: C, 62.76;
H, 4.71.
8-Phenyl-3H-1,2-benzoxathiepine 2,2-dioxide (21)
Compound 21 was prepared according to the general procedure from 8-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (20) (0.25 g; 0.91 mmol) phenylboronic acid (0.17 g; 1.36 mmol), K3PO4 (0.39 g;
1.82 mmol) and Pd(PPh3)4 (105 mg; 0.091 mmol) as yellowish solid
(109 mg; 44%). Mp 103–104C. IR (film, cm1)max¼ 1376 (S ¼ O),
1177 (S¼ O). 1H NMR (400 MHz, CDCl
3) d¼ 4.08 (dd, 2H, J ¼ 6.1,
1.2 Hz), 5.94–6.01 (m, 1H), 6.88–6.93 (m, 1H), 7.36–7.43 (m, 2H), 7.44–7.50 (m, 2H), 7.55–7.63 (m, 4H) ppm. 13C KMR (100 MHz, CDCl3) d¼ 51.6, 119.2, 121.3, 125.7, 126.8, 127.2, 128.5, 129.2,
131.3, 132.5, 138.9, 144.0, 148.1 ppm. Anal. Calcd for C15H12O3S: C,
8–(4-Methoxyphenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (22)
Compound 22 was prepared according to the general procedure from 8-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (20) (0.25 g; 0.91 mmol) 4-methoxyphenylboronic acid (0.21 g; 1.36 mmol), K3PO4 (0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg; 0.091 mmol) as
yellowish solid (121 mg; 44%). Mp 142–143C. IR (film, cm1) max¼ 1369 (S ¼ O), 1177 (S ¼ O), 1164 (S ¼ O).1H NMR (400 MHz,
CDCl3) d¼ 3.86 (s, 3H), 4.07 (dd, 2H, J ¼ 6.1, 1.1 Hz), 5.92–5.99 (m,
1H), 6.88 (d, 1H, J¼ 11.1 Hz), 6.97–7.02 (m, 2H), 7.32–7.36 (m, 1H), 7.50–7.58 (m, 4H) ppm.13C NMR (100 MHz, CDCl3) d¼ 51.6, 55.5,
114.6, 118.8, 120.6, 125.2, 126.1, 128.3, 131.3, 132.6, 143.6, 148.2, 160.1 ppm. Anal. Calcd for C16H14O4S: C, 63.56; H, 4.67. Found: C,
63.20; H, 4.69.
8–(4-Fluorophenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (23)
Compound 23 was prepared according to the general procedure from 8-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (20) (0.25 g; 0.91 mmol) (4-fluorophenyl)boronic acid (0.19 g; 1.36 mmol), K3PO4
(0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg; 0.091 mmol) as white
solid (108 mg; 41%). Mp 111–112C. IR (film, cm1) max¼ 1371
(S¼ O), 1168 (S ¼ O). 1H NMR (400 MHz, CDCl3) d¼ 4.08 (dd, 2H, J¼ 6.1, 1.2 Hz), 5.94–6.01 (m, 1H), 6.90 (d, 1H, J ¼ 11.0 Hz), 7.12–7.19 (m, 2H), 7.35–7.40 (m, 1H), 7.50–7.53 (m, 2H), 7.54–7.60 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3) d¼ 51.7, 116.2 (d, J¼ 21.6 Hz), 119.3, 121.2, 125.6, 126.9, 128.9, 129.0, 131.5, 132.4, 135.0 (d, J¼ 3.3 Hz), 142.9, 148.1, 163.2 (d, J ¼ 248.0 Hz) ppm. Anal. Calcd for C15H11FO3S: C, 62.06; H, 3.82. Found: C, 62.04; H, 3.86.
8–(4-(Trifluoromethyl)phenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (24)
Compound 24 was prepared according to the general procedure from 8-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (20) (0.25 g; 0.91 mmol) (4-(trifluoromethyl)phenyl)boronic acid (0.26 g; 1.36 mmol), K3PO4 (0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg;
0.091 mmol) as white solid (142 mg; 46%). Mp 121–122C. IR (film, cm1) max¼ 1366 (S ¼ O), 1324 (S ¼ O), 1172 (S ¼ O).
1 H NMR (400 MHz, CDCl3) d¼ 4.11 (dd, 2H, J ¼ 6.1, 1.2 Hz), 5.97–6.04 (m, 1H), 6.90 (d, 1H, J¼ 11.2 Hz), 7.40–7.44 (m, 1H), 7.55–7.60 (m, 2H), 7.70–7.75 (m, 4H) ppm.13C NMR (100 MHz, CDCl3) d¼ 51.8, 119.7, 121.6, 124.2 (q, J¼ 273.0 Hz), 125.9, 126.2 (q, J ¼ 3.8 Hz), 127.6, 127.8, 130.5 (q, J¼ 32.9 Hz), 131.7, 132.2, 142.3, 142.4, 148.1 ppm. Anal. Calcd for C16H11F3O3S: C, 56.47; H, 3.26. Found: C, 56.23;
H, 3.23.
8–(4-(Ethoxycarbonyl)phenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (25)
Compound 25 was prepared according to the general procedure from 8-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (20) (0.25 g; 0.91 mmol) (4-(ethoxycarbonyl)phenyl)boronic acid (0.26 g; 1.36 mmol), K3PO4 (0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg;
0.091 mmol) as white solid (119 mg; 38%). Mp 151–152C. IR (film, cm1) max¼ 1703 (C ¼ O), 1366 (S ¼ O), 1175 (S ¼ O). 1H NMR
(400 MHz, CDCl3) d¼ 1.42 (t, 3H, J ¼ 7.1 Hz), 4.10 (dd, 2H, J ¼ 6.1,
1.2 Hz), 4.41 (q, 2H, J¼ 7.1 Hz), 5.96–6.03 (m, 1H), 6.90 (d, 1H, J¼ 11.2 Hz), 7.39–7.43 (m, 1H), 7.57–7.62 (m, 2H), 7.65–7.70 (m, 2H), 8.11–8.16 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3)
d¼ 14.5, 51.7, 61.3, 119.6, 121.5, 125.9, 127.1, 127.7, 130.4, 131.6, 132.3, 142.7, 143.0, 148.1, 166.3 ppm. Anal. Calcd for C18H16O5S: C,
62.78; H, 4.68. Found: C, 62.50; H, 4.70.
9- Phenyl-3H-1,2-benzoxathiepine 2,2-dioxide (30)
Compound 30 was prepared according to the general procedure from 9-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (29) (0.25 g; 0.91 mmol) phenylboronic acid (0.17 g; 1.36 mmol), K3PO4 (0.39 g;
1.82 mmol) and Pd(PPh3)4 (105 mg; 0.091 mmol) as white solid
(104 mg; 42%). Mp 135–136C. IR (film, cm1)max¼ 1370 (S ¼ O),
1162 (S¼ O). 1 H NMR (400 MHz, CDCl3) d¼ 4.08 (dd, 2H, J ¼ 5.8, 1.3 Hz), 5.87–5.94 (m, 1H), 6.85–6.90 (m, 1H), 7.29 (dd, 1H, J ¼ 7.6, 1.8 Hz), 7.35–7.42 (m, 2H), 7.43–7.49 (m, 3H), 7.51–7.55 (m, 2H) ppm. 13C KMR (100 MHz, CDCl3) d¼ 52.1, 118.9, 127.1, 128.1, 128.5, 128.6, 129.6, 130.5, 132.1, 132.5, 136.3, 136.5, 144.7 ppm. Anal. Calcd for C15H12O3S: C, 66.16; H, 4.44. Found: C, 66.15;
H, 4.46.
9–(4-Methoxyphenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (31)
Compound 31 was prepared according to the general procedure from 9-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (29) (0.25 g; 0.91 mmol) 4-methoxyphenylboronic acid (0.21 g; 1.36 mmol), K3PO4 (0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg; 0.091 mmol) as
white solid (110 mg; 40%). Mp 113–114C. IR (film, cm1) max¼
1369 (S¼ O), 1181 (S ¼ O), 1154 (S ¼ O).1H NMR (400 MHz, CDCl 3) d¼ 3.85 (s, 3H), 4.08 (dd, 2H, J ¼ 5.8, 1.3 Hz), 5.86–5.94 (m, 1H), 6.84–6.89 (m, 1H), 6.97–7.02 (m, 2H), 7.23–7.27 (m, 1H), 7.34 (t, 1H, J¼ 7.6 Hz), 7.42 (dd, 1H, J ¼ 7.6, 1.8 Hz), 7.45–7.50 (m, 2H) ppm. 13C NMR (100 MHz, CDCl 3) d¼ 52.0, 55.4, 114.0, 118.9, 127.1, 128.6, 128.7, 130.1, 130.8, 132.0, 132.6, 136.2, 144.7, 159.5 ppm. Anal. Calcd for C16H14O4S: C, 63.56; H, 4.67. Found: C, 63.58;
H, 4.70.
9–(4-Fluorophenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (32)
Compound 32 was prepared according to the general procedure from 9-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (29) (0.25 g; 0.91 mmol) (4-fluorophenyl)boronic acid (0.19 g; 1.36 mmol), K3PO4
(0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg; 0.091 mmol) as white
solid (103 mg; 39%). Mp 130–131C. IR (film, cm1) max¼1370
(S¼ O), 1154 (S ¼ O). 1H NMR (400 MHz, CDCl 3) d¼ 4.08 (dd, 2H, J¼ 5.8, 1.3 Hz), 5.88–5.95 (m, 1H), 6.85–6.90 (m, 1H), 7.10–7.18 (m, 2H), 7.30 (dd, 1H, J¼ 7.5, 2.0 Hz), 7.37 (t, 1H, J ¼ 7.5 Hz), 7.41 (dd, 1H, J¼ 7.5, 2.0 Hz), 7.47–7.53 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3) d¼ 52.1, 115.5 (d, J ¼ 21.6 Hz), 119.1, 127.2, 128.7, 130.6, 131.3, 131.4, 132.0, 132.3 (d, J¼ 3.3 Hz), 132.5, 135.6, 144.7, 162.8 JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 249
(d, J¼ 247.0 Hz) ppm. Anal. Calcd for C15H11FO3S: C, 62.06; H, 3.82.
Found: C, 62.05; H, 3.84.
9–(4-(Trifluoromethyl)phenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (33)
Compound 33 was prepared according to the general procedure from 9-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (29) (0.25 g; 0.91 mmol) (4-(trifluoromethyl)phenyl)boronic acid (0.26 g; 1.36 mmol), K3PO4 (0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg;
0.091 mmol) as white solid (136 mg; 44%). Mp 115–116C. IR (film, cm1) max¼ 1333 (S ¼ O), 1166 (S ¼ O).
1 H NMR (400 MHz, CDCl3) d¼ 4.10 (dd, 2H, J ¼ 5.8, 1.3 Hz), 5.90–5.97 (m, 1H), 6.86–6.91 (m, 1H), 7.35 (dd, 1H, J ¼ 7.0, 2.6 Hz), 7.38–7.45 (m, 2H), 7.62–7.67 (m, 2H), 7.70–7.74 (m, 2H) ppm.13C NMR (100 MHz, CDCl3) d¼ 52.2, 119.2, 124.5 (q, J ¼ 273.0 Hz), 125.5 (q, J ¼ 3.8 Hz), 127.3, 128.9, 130.0, 130.2 (q, J¼ 32.0 Hz), 131.3, 131.9, 132.3, 135.2, 140.0 (q, J¼ 1.5 Hz), 144.6 ppm. Anal. Calcd for C16H11F3O3S: C,
56.47; H, 3.26. Found: C, 56.21; H, 3.29.
9–(4-(Ethoxycarbonyl)phenyl)-3H-1,2-benzoxathiepine 2,2-dioxide (34)
Compound 34 was prepared according to the general procedure from 9-bromo-3H-1,2-benzoxathiepine 2,2-dioxide (29) (0.25 g; 0.91 mmol) (4-(ethoxycarbonyl)phenyl)boronic acid (0.26 g; 1.36 mmol), K3PO4 (0.39 g; 1.82 mmol) and Pd(PPh3)4 (105 mg;
0.091 mmol) as white solid (113 mg; 36%). Mp 105–106C. IR (film, cm1)max¼ 1714 (C ¼ O), 1375 (S ¼ O), 1157 (S ¼ O). 1 H NMR (400 MHz, CDCl3) d¼ 1.41 (t, 3H, J ¼ 7.1 Hz), 4.08 (dd, 2H, J¼ 5.8, 1.1 Hz), 4.40 (q, 2H, J ¼ 7.1 Hz), 5.89–5.97 (m, 1H), 6.88 (d, 1H, J¼ 11.4 Hz), 7.31–7.46 (m, 3H), 7.58–7.63 (m, 2H), 8.11–8.16 (m, 2H) ppm. 13C NMR (100 MHz, CDCl 3) d¼ 14.5, 52.1, 61.1, 119.2, 127.2, 128.8, 129.6, 129.7, 130.1, 131.1, 131.8, 132.4, 135.6, 140.9,
144.6, 166.5 ppm. Anal. Calcd for C18H16O5S: C, 62.78; H, 4.68.
Found: C, 62.28; H, 4.69.
2.2. CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2hydration activity15. Phenol red (at a
concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buf-fer and 20 mM Na2SO4 (for maintaining constant the ionic strength),
following the initial rates of the CA-catalysed CO2hydration reaction
for a period of 10 100 s. The CO2 concentrations ranged from 1.7
to 17 mM for the determination of the kinetic parameters and inhib-ition constants. For each inhibitor, at least six traces of the initial 5 10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled deionised water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6 h at room temperature prior to assay in order to allow for the formation of the E I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng Prusoff equation, as reported earlier16–19, and represent the mean from at least three different determinations. All CA isoforms were recombin-ant ones obtained in-house as reported earlier19,20.
3. Results and discussion
3.1. ChemistryThe synthesis of desired compounds is partly based on the strat-egy previously developed by our groups10. The synthesis of 7-aryl 3H-1,2-benzoxathiepine 2,2-dioxides starts with the iodination of salicylaldehyde (1) by iodine monochloride and corresponding iodo derivative 2 was isolated in good yield (Scheme 1)11. Under Wittig reaction conditions aldehyde 2 was converted to olefin 3, which was treated by sulphonyl chloride 4 thus providing bis-ole-fin 5 in 83% yield. To obtain the key intermediate 7, the ring clos-ure in compound 5 was performed in olefin metathesis
Scheme 1. Reagents and conditions for the preparation of derivatives8–12: (i) ICl, AcOH, 40C, 24 h, 84%; (ii) KOtBu, CH3P(C6H5)3Br, THF, RT, 18 h, 83%; (iii) NEt3, CH2Cl2, 0C to RT, 4 h, 83%; (iv) toluene, 70C, 4 h, 89%; (v) Ar-B(OH)2, Pd(PPh3)4, K3PO4, toluene/H2O, 100C, 16 h.
conditions, using Ru-catalyst 6. The key intermediate 7 was reacted with a series of aryl boronic acids under Suzuki reaction conditions and the desired 7-aryl 3H-1,2-benzoxathiepine 2,2-diox-ides 8–12 were isolated in acceptable yields (44–66%) (Scheme 1). In an attempt to prepare 6-aryl 3H-1,2-benzoxathiepine 2,2-diox-ides, the commercially available bromo salicylaldehyde 13 was first converted to olefin 14 under Wittig reaction conditions, followed by treatment with sulphonyl chloride 4, thus providing bis-olefin 15 for olefin metathesis ring closure reaction (Scheme 2). Utilisation of the Ru-catalyst 6 as described above did not provide the formation of the desired key intermediate 6-bromo 3H-1,2-benzoxathiepine 2,2-dioxide (16) even at prolonged reaction times. By doubling cata-lyst 6 amount (10 mol%) only traces of compound 16 were observed after 40 h. No product formation was observed also when using Schrock and Schrock–Hoveyda Mo-catalysts. Probably olefin metathesis ring closure reaction did not take place due to sterical constraints due to the bulky Br atom at 3-positon of bis-olefin 15.
The synthesis of 8-bromo intermediate 20 was started from commercially available aldehyde 17, when under Wittig reaction conditions olefin 18 was obtained, which was thereafter treated with sulphonyl chloride 4 and provided the bis-olefin 19 in good yield (Scheme 3). Ru-catalysed olefin metathesis afforded the key intermediate 20 which in turn, by reaction with a series of aryl boronic acids under Suzuki reaction condition, provided the desired compounds 21–25.
The same strategy was successfully utilised for the synthesis of a series of 9-aryl 3H-1,2-benzoxathiepine 2,2-dioxides starting by
the treatment of aldehyde 26 with methyltriphenylphosphonium bromide under Wittig reaction conditions (Scheme 4). The obtained phenol 27 was reacted with sulphonyl chloride 4 and ring closure of isolated 28 was successfully performed in Ru-cata-lysed olefin metathesis conditions, providing bromide 29. Further reaction of compound 9 with aryl boronic acids provided the desired derivatives 30–34 in moderate yields.
3.2. Carbonic anhydrase inhibition
The obtained homosulfocoumarins 7–34 were investigated for their CA inhibitory properties by using a stopped-flow CO2
hydrase assay15 and four human CA isoforms (hCA I, II, IX and XII) known to be drug targets1(Table 1).
The following structure-activity relationship (SAR) can be observed from the inhibition data ofTable 1.
(i) as the previously reported homosulfocoumarins10and similar to sulfocoumarins7–9, also the derivatives reported here did not significantly inhibit the cytosolic isoforms hCA I and II, unlike the sulphonamide acetazolamide (used as standard CAI), which has a very good affinity (in the nanomolar range) for hCA II and a micromolar one for hCA I (Table 1).
(ii) the transmembrane, tumour-associated isoforms hCA IX and XII were effectively inhibited by derivatives 7–29 reported here (in the low – medium nanomolar arneg) and were poorly inhibited, in the micromolar range by the 9-substituted-homosulfocoumarins 30–34 (KIs in the range of 16.4–60.9 mM against hCA IX and >100 mM
Scheme 2. Reagents and conditions: (i) KOtBu, CH3P(C6H5)3Br, THF, RT, 18 h, 82%; (ii)4, NEt3, CH2Cl2, 0C to RT, 4 h, 66%; (iii) a)6 (5 mol% and 10 mol%), toluene, 70C, 40 h, 0%; b) Schrock catalyst [Mo] (10 mol%), toluene, 70C, 16 h, 0%; c) Schrock–Hoveyda [Mo] (10 mol%), toluene, 70C, 16 h, 0%;
Scheme 3. Reagents and conditions: (i) KOtBu, CH3P(C6H5)3Br, THF, RT, 18 h, 76%; (ii)4, NEt3, CH2Cl2, 0C to RT, 4 h, 54%; (iii)6, toluene, 70C, 4 h, 90%; (iv) Ar-B(OH)2, Pd(PPh3)4, K3PO4, toluene/H2O, 100C, 16 h.
against hCA XII). Thus, although weak inhibitors, these sulfocoumarins are anyhow highly selective for the inhibition of hCA IX, whereas their activity against hCA I, II and XII is absent (Table 1). As already anticipated above, the most important factors associated with CA IX/ XII inhibitory activity are the position and the nature of the moieties present on the six-membered ring of the homosulfocoumarin. Indeed, for 9-substituted derivatives, the presence of bulky, substi-tuted aryls as in 30–34 leads to low activity, as mentioned above. Only the 9-bromo-derivative 29 had a medium potency inhibitory action against the two isoforms, with KIs in the range of 754.8 –
3824 nM. On the contrary, the 8-substituted derivatives 20–25 showed a much better inhibitory power against both isoforms, being generally more potent than the corresponding 7-substituted deriva-tives 7–12. Indeed, for the 7-substituted homosulfocoumarins the KIs
were in the range of 66.2– 620.8 nM against hCA IX and of 455.5 –
2934 nM against hCA XII. On the contrary, for the 8- substituted homosulfocoumarins, the KIs were in the range of 44.0 – 104.8 nM
against hCA IX and in the range of 77.9 – 473.2 nM for hCA XII (Table 1). The 8–(4-trifluoromethyl)phenyl-substituted homosulfocou-marin 24 was the most effective hCA IX inhibitor (potency in the same range as AAZ), whereas the corresponding 4-fluorophenyl derivative 23 was the best hCA XII inhibitor in the new series of com-pounds investigated here but it was an order of magnitude less effective compared to acetazolamide.
4. Conclusions
A new series of homosulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) possessing various moieties in the 7, 8 or 9 position of the heterocylic ring were prepared by original procedures and investigated for the inhibition of four physiologically relevant CA isoforms, hCA I, II, IX and XII. The 8-substituted homosulfocoumarins were the most effective hCA IX/XII inhibitors followed by the 7-sub-stituted derivatives, whereas the substitution pattern in position 9 led to less effective inhibitors for these transmembrane, tumour-associated isoforms. The cytosolic isoforms hCA I and II were not inhibited by these compounds, similar to the sulfocoumarins/cou-marins investigated earlier. As hCA IX and XII are validated anti-tumour targets5, with one sulphonamide (SLC-0111) in Phase Ib/II clinical trials, finding derivatives with a better selectivity for inhibit-ing the tumour-associated isoforms over the cytosolic ones, as the homosulfocoumarins reported here, is of crucial importance.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by ERA.Net RUS plus joint programme pro-ject THIOREDIN (State Education Development Agency of Republic of Latvia) [grant number RUS_ST2017-309] and the Russian Foundation for Basic Research [grant number 18–515-76001].
ORCID
Claudiu T. Supuran http://orcid.org/0000-0003-4262-0323
Scheme 4. Reagents and conditions: (i) KOtBu, CH3P(C6H5)3Br, THF, RT, 18 h, 80%; (ii)4, NEt3, CH2Cl2, 0C to RT, 4 h, 86%; (iii)6, toluene, 70C, 4 h, 78%; (iv) Ar-B(OH)2, Pd(PPh3)4, K3PO4, toluene/H2O, 100C, 16 h.
Table 1. Inhibition data of human CA isoforms CA I, II, IX and XII with 3H-1,2-benzoxathiepines 2,2-dioxide7–34 using AAZ as a standard drug.
Cmpd 7 / 8 / 9 R KI(nM) CA I CA II CA IX CA XII 7 7 I >100 mM >100 mM 66.2 455.5 8 7 H >100 mM >100 mM 654.8 1376 9 7 OCH3 >100 mM >100 mM 407.6 2934 10 7 F >100 mM >100 mM 330.8 890.5 11 7 CF3 >100 mM >100 mM 221.4 4017 12 7 CO2CH2CH3 >100 mM >100 mM 620.8 2398 20 8 Br >100 mM >100 mM 47.5 132.9 21 8 H >100 mM >100 mM 104.8 473.2 22 8 OCH3 >100 mM >100 mM 63.1 168.6 23 8 F >100 mM >100 mM 95.2 77.9 24 8 CF3 >100 mM >100 mM 44.0 247.8 25 8 CO2CH2CH3 >100 mM >100 mM 79.8 289.3 29 9 Br >100 mM >100 mM 754.8 3824 30 9 H >100 mM >100 mM 21.1mM >100 mM 31 9 OCH3 >100 mM >100 mM 60.9mM >100 mM 32 9 F >100 mM >100 mM 33.7mM >100 mM 33 9 CF3 >100 mM >100 mM 47.1mM >100 mM 34 9 CO2CH2CH3 >100 mM >100 mM 16.4mM >100 mM AAZ – – 250 12 25 5.7
Mean from three different assays, by a stopped flow technique (errors were in the range of ± 5–10% of the reported values).
References
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